CN1216997C - Cotton fiber reversible glycosylated proteinase gene and protein coded therewith - Google Patents

Abstract

The present invention relates to a cotton fiber reversible glycosylated proteinase gene and protein coded by the gene. The gene for coding cotton reversible glycosylated protein of the present invention has the nucleotide sequence of sequence-1, which is composed of 1331 basic groups and contains an integral open reading frame named as GhRGP1. The cotton reversible glycosylated proteinase of the present invention is composed of 359 amino acid residues and is sequence-2 in the sequence list, with the theoretical molecular weight of 40.78Da and the isoelectric point of 6.01. The discovery and clone of the cotton reversible glycosylated protein gene cause the accumulation of polysaccharide substances in the control of cotton fibers by means of genetic engineering to be possible and lay a foundation for the cultivation of transgenic cotton with high yield and good quality.

Description

Cotton fiber reversible glycosylated proteinase gene and encoded protein matter thereof

Technical field

The present invention relates to a kind of gene of cotton and coded protein thereof.

Background technology

Plant cell wall polysaccharides is the abundantest in the world biomacromolecule, and their synthetic is under the catalysis of membrane bound enzyme, utilizes nucleosides sugar to form polysaccharide as glycosyl donor.Most important cell wall polysaccharides is hemicellulose and Mierocrystalline cellulose, the former is by the localized glucan synthase I of golgi body (GSI) catalysis, the latter is by the localized glucan synthase II of plasmalemma (GSII) catalysis, and GSII comprises Mierocrystalline cellulose synthetic enzyme and callose synthetic enzyme.

Because membrane bound enzyme is very unstable, extremely responsive to stain remover, lose activity easily, very difficult separation and purification obtains activated enzyme, has seriously limited going deep into of research for a long time.Up to 1991, when Dhugga etc. (1997) synthesize polysaccharide at the Golgi membrane of research pea, identify the albumen dimer of a 41kD, can be by radiolabeled UDP-Glu glycosylation, this glycosylation can reversibly be competed combination by nonradioactive labeling's UDP-Glu, UDP-Xyl, UDP-Gal, thereby with this protein called after reversible glycosylated proteinase (reversiblyglycosylated polypeptide, RGP).The glycosylation condition of pea PsRGP is identical with the GSI reactive conditions.In order to study the effect of RGP in polysaccharide is synthetic, Dhugga etc. (1997) purifying PsRGP, the preparation antiserum(antisera).By immunoscreening pea cDNA expression library, cloned the cDNA of PsRGP.Their PsRGP that studies show that has the enzyme activity, and gold-marking immunity is located the face in the trans of golgi body.Delgado etc. (1998) have cloned AtRGP1 from the model plant Arabidopis thaliana, obtained to have the gene with the PsRGP identity function, be a monomeric protein but difference is AtRGP1, and PsRGP are two body proteins.

Summary of the invention

The objective of the invention is to clone the gene of cotton reversible glycosylated proteinase, and the cotton reversible glycosylated proteinase by this genes encoding is provided.

The gene of cotton reversible glycosylated proteinase of the present invention contains a complete open reading frame by 1331 based compositions, is No. 1 sequence in the sequence table, called after GhRGP1.

The gene of the reversible glycated protein enzyme of cotton is made up of non-coding region, the reading frame coding region of 1080bp and the 3 ' non-coding region of 201bp of 5 ' end 50bp.The initiator codon of opening code-reading frame is ATG, and terminator codon is TGA.

Cotton reversible glycosylated proteinase of the present invention is made up of 359 amino-acid residues, is the sequence in the sequence table 2.

The theoretical molecular of cotton reversible glycosylated proteinase is 40.78Da, and iso-electric point is 6.01.

The cotton reversible glycosylated proteinase is the embrane-associated protein of solubility, is positioned the trans face of golgi body in the cell, participates in the biosynthesizing of cell wall polysaccharides.Reversible glycosylated proteinase can be reversibly by UDP-Glu, UDP-Xyl, the glycosylation of UDP-Gal institute, and its glycosylation ratio is UDP-Glu :-Xyl :-Gal10: 7: 3, the moiety of this ratio and xyloglucan was closely similar.

As shown in Figure 1, the GhRGP1 of the cDNA sequence encoding of total length 1331bp of the present invention and the RGP of other plant are carried out homology relatively, wherein, Gh is a cotton, and Ps is a pea, At is an Arabidopis thaliana, Os is a paddy rice, and Ta is a wheat, and Zm is a corn, Vu is a cowpea, and the result shows that the homology of cotton reversible glycosylated proteinase aminoacid sequence and corn, wheat, pea, cowpea, paddy rice and Arabidopis thaliana RGP is respectively 86%, 85%, 85%, 84%, 84% and 78%.With molecular biology software (pSORT, http://psort.nibb.ac.jp) forecast analysis GhRGP1 lacks signal peptide, hydrophobicity analysis shows that GhRGP1 does not have the diaphragm area of striding, and is soluble proteins, and this situation with the homologous protein that the middle separation and purification of other plant (pea and Arabidopis thaliana) is arrived is consistent.GhRGP1 has the conserved residues (D is positioned at 44) and the structural domain (DXD is positioned at 102) of non-processed-type glycosyltransferase, is UDP-sugar potential binding site, and saccharification residue (R) is positioned at 150.These conserved residues are different with existing RGP with structural domain and saccharification residue position, so new plant RGP gene of GhRGP1 cDNA coding, can play nucleosides sugar transport body or intermediate in the biosynthesizing of cell wall polysaccharides.

Experiment shows that GhRGP1 is single copy gene, may contain intron in allotrtraploid AD genome.Concrete experimentation is, from the cotton variety middle cotton extract genomic dna 12 blades that stretching.There is not the restriction enzyme BamHI of cleavage site with GhRGP1 gene inside, EcoRI, EcoRV, KpnI and XbaI be enzymolysis 20 μ g genomic dnas respectively, after 0.7% agarose gel electrophoresis separates, DNA are gone to nylon membrane Hybond-N ⁺On.(Pierce, method USA) after 0.5 hour, added the GhRGP1 cDNA probe of mark at 55 ℃ of following prehybridizations with this film, in 55 ℃ of hybridization 2 hours to press Northern2Southern HRP marker detection test kit.The hybridization back is with containing the film washing liquid of 0.1%SDS and 2 * SSC 55 ℃ of following rinsings 3 times, each 5 minutes; And then wash film 3 times in room temperature, each 5 minutes with the film washing liquid of 2 * SSC; Add colour developing liquid at last, room temperature was placed 10 minutes, discarded colour developing liquid, sudden and violent X-ray sheet.The result as shown in Figure 2, wherein, B:BamHI; EI:EcoR I; K:Kpn I; EV:EcoRV all shows 1 band after the enzymolysis product hybridization of 4 kinds of restriction endonucleases, illustrates that GhRGP1 is single copy in allotrtraploid AD genome; And the variation that 2 carrying means may contain this restriction enzyme site between intron or allotrtraploid AD genome has appearred in the XbaI enzyme cutting product.

In order to identify the tissue distribution of GhRGP1 gene, be that material carries out Northern hybridization with fiber, stem, root, leaf, flower, ovule and seed.Hybridizing method is identical with the Southern hybrid method.The result shown in Fig. 3-A and Fig. 3-B, wherein, Fb among Fig. 3-A: the fiber of growing 10 days; Rt: young root; St: stem; Lf: leaf; Fl: flower; Ov: ovule; Sd: the seed of growing 30 days.The fate that fiber was grown after numeral was bloomed among Fig. 3-B.As can be seen from the figure, GhRGP1 is strong predominant expression in fibrocyte, secondly in young root, also have than high expression level, spend, ovule and the low scale of seed reach, and express at stem and Ye Zhongwu.The great expression of this hint GhRGP1 is to get in touch with the quick growth phase of cell or tissue.

The discovery of the reversible glycosylated protein gene of cotton and clone make the accumulation by polysaccharide material in the genetic engineering means regulation and control cotton fiber become possibility, and are that the transgene cotton of cultivating high yield and high quality is laid a good foundation.

The invention will be further described below in conjunction with embodiment.

Description of drawings

The proteic homology contrast of Fig. 1 cotton RGP1 and other plant RGP.

Fig. 2 is the Southern hybridization of GhRGP1.

Fig. 3-A is the Northern hybridization of GhRGP1 mRNA, shows the distribution characteristics of GhRGP1 mRNA at the cotton different tissues.

Fig. 3-B is the Northern hybridization of GhRGP1 mRNA, shows the expression characteristic of GhRGP1 mRNA in the cotton fiber development process.

Fig. 4-A is 5 ' the RACE-PCR product agarose gel electrophoresis result of GhRGP1.

Fig. 4-B is that the enzyme of pGEM-T easy-GhRGP1 is cut evaluation.

Fig. 5 is the expression vector pQE-30-GhRGP1 of GhRGP1.

Fig. 6 is the expression of pQE-30-GhRGP1 in M15.

Embodiment

Embodiment 1, fluorescence difference show clone's fiber specifically expressing cDNA fragment

Good Cultivar middle cotton institute's 12 conventional seed plantings of upland cotton (Gossypium hirsutum) and field management, bloom the same day to the cotton boll mark of listing, and the different number of days after blooming is gathered cotton buds and bolls, strip ovule and fiber, leaf, stem and flower (bract, petal and stamen) are gathered from ripe plant, root is 3 days a radicle behind the seed germination, and all material after the quick-frozen, is stored in-70 in liquid nitrogen.Extract cotton different tissues and fibrocellular total RNA, grow 10 days fiber and total RNA of seed and be used for the demonstration of fluorescence difference.

1, the extraction of total RNA

Extract damping fluid:

Sodium Tetraborate pH9.0 0.2M

EGTA 30mM

SDS 1.0％

DOC 1.0％

PVP-40 2.0％

DTT 0.1M

Step:

(1) 80 ℃ of preheating extracting solution 10ml.

(2) cotton tissue 2g, the liquid nitrogen grinding powdered.

(3) powder is dropped into the extracting solution 10ml (5ml/1g) of preheating, 2min homogenizes.

(4) with after 4 layers of sterile gauzes filtration, add 400l Proteinase K mixing, 37 ℃, 1～1.5hr, 200rpm.

(5) add 0.8ml 2M KCl, place 1hr on ice; 4 ℃, 12000rpm, 20min.

(6) the 10M LiCl of adding 1/4 volume, to final concentration 2M, 4 ℃ of precipitations are spent the night.

(7) 12000rpm, 4 ℃, centrifugal 20min abandons supernatant.

(8) with cold 2M LiCl washing 2 times, each centrifugal 10min of 10000rpm.

(9) with 800l TE dissolution precipitation, the centrifugal 5min of 10000rpm gets supernatant.

(10) add 2M KAc to final concentration 0.2M, place 15min on ice; The centrifugal 10min of 10000rpm gets supernatant.

(11) add 2.5 times of volume of ethanol ,-20 ℃ of precipitation 5-8hr.

(12) 12000rpm, in 4 ℃ of centrifugal 20min, 80% washing with alcohol 1 time is drained, and is dissolved among the 100ul TE, in-70 ℃ of preservations.Take a morsel and carry out electrophoresis detection, and measure OD value, calculating concentration.

2, fluorescence difference shows acquisition cotton RGP3 ' fragment

Difference is presented on Genomyx (Beckman) instrument carries out, and uses the FloroDD test kit of the said firm to operate.From the synthetic cDNA of total RNA reverse transcription, and is template with cDNA with ThermoScript II Superscript RT (Gibco), selects for use 3 pairs of primers to carry out PCR.Anchor primer is by the T7 promoter sequence of 17bp, the polyT of 12bp and 2 variable based compositions, and random primer is made up of the TM13 reverse sequence (M13r) of 16bp and the stochastic sequence of 10bp.Anchor primer 5 ' end fluorescent mark, random primer is mark not.This tests used anchor primer is AP7 (5 ' T7dT ₁₂CG3 ') and AP9 (5 ' T7dT ₁₂AC3 '), random primer is ARP19 (5 ' M13r-TTTTGGCTCC3 ') and ARP20 (5 ' M13r-TCGATACAGG3 '), and combination of primers is AP7/ARP19, AP7/ARP20, AP9/ARP19.The PCR product is electrophoresis on high resolving power, 5.6% sex change glue, scans behind the dried glue.Take the special band that occurs in the fiber, and in rigorous condition (94 ℃ of 2min, a circulation; 94 30sec, 60 30sec, 72 2min, 30 circulations; 72 7min, a circulation.) following secondary PCR, reclaim these cDNA fragments, with its be cloned into the TA carrier (pGEM-T Easy, Promega) on, on 377 instrument, check order.

Fluorescence difference shows the candidate segment that obtains specifically expressing in 16 fibers altogether, and one of them (F12) has very high homology with the RGP3 ' end of known array, and the Northern dot blot confirms this fragment strong predominant expression in fibrocyte.Confirm, obtained the 3 ' fragment of cotton RGP.

Embodiment 2, have the structure in the cotton fiber cDNA library of double-end

From 10 days the total RNA of cotton fiber of back growth of blooming, adopt the PolyATtract System1000 separating mRNA of Promega, with the synthetic cDNA of the Marathon cDNA test kit of Clontech, connect joint again, be built into the cotton fiber cDNA library that two ends have joint.

1, the separation of cotton fiber mRNA

With the PolyATtract System of Promega company 1000 separating mRNA from the total RNA that grows 10 days fibers, the concrete operations step is as follows:

(1) get total RNA 750 μ g, volume 500 μ l add the biotinylated oligomerizations of 3 μ l (dT) nucleotide probe, mixing gently, and in 70 ℃ of sex change 5min, room temperature is placed, cooling balance 10min.

(2) wash SA-PMPs magnetic ball 3 times with 1ml SSC (0.5 *), last resuspended magnetic ball is in 500ul SSC (0.5 *).And suspension is moved in the mRNA User pipe of this test kit outfit, adsorb SA-PMPs magnetic ball particle once more with magnet, the careful suction removed SSC solution.Repeated washing 2 times.

(3) with the mRNA/ probe reaction thing of renaturation, join in the good SA-PMPs magnetic ball of washing, mixing gently, room temperature is placed 10min.

(4) pounce on to obtain with magnet stand and move the SA-PMPs/mRNA/ probe complex, careful supernatant discarded.

(5) with 1ml SSC (0.5 *) washing 3 times.Centrifugal termination back carefully moves into supernatant to be contained in the tapered tube of 2ml SA-PMPs magnetic ball, the mixing that turns upside down, and after room temperature is placed 2min, with magnet absorption SA-PMPs magnetic ball, until the solution clarification, and careful supernatant discarded.

(6) use the ultrapure water of 600l resuspended, pounce on magnet stand and obtain the SA-PMPs/ probe complex, carefully draw supernatant.

(7) supernatant that will contain mRNA is transferred in the new pipe, adds 1/10 volume 3M NaAc (pH5.2) and equal-volume Virahol, and-20 ℃ of placements are spent the night behind the mixing.

(8) 4 ℃ 12, the centrifugal 10min of 000rpm, supernatant discarded precipitates with 70% ethanol centrifuge washing.

(9) the vacuum-drying precipitation is about 15 minutes, and with the ultrapure water suspension precipitation of an amount of volume ,-70 ℃ of preservations are standby.

2, the structure in cDNA library

Schedule of operation according to the Marathon cDNA of Clontech company test kit, be that the template reverse transcription becomes the first chain cDNA with cotton fiber mRNA earlier, be synthetic second chain of template with the first chain cDNA then, more double-stranded cDNA two connected joint and constitute the cDNA library that two ends have joint.The concrete operations step is as follows:

A, synthetic cDNA first chain

(1) in the 0.5ml Eppendorf tube, add following reactants:

mRNA(1g) 4l

CDNA synthesizes oligomerization d (T) primer (10M) 1l

Mixing is also centrifugal a little gently, 70 ℃ of sex change 5min.

(2) continue in the 0.5ml Eppendorf tube, to add following reactants:

5 * the first chain damping fluid 2l

DNTP mixture (10mM) 1l

AMV ThermoScript II (20U/l) 1l

Add water cumulative volume is supplied 10l

Gently mixing and centrifugal a little after, in 42 ℃ of airbaths 1 hour with synthetic cDNA.Then reaction tubes is placed on ice to end first chain synthesis reaction.

B, synthetic cDNA second chain

(1) in the first chain reaction pipe, add following reactants:

ddH ₂O 48.4l

Second chain damping fluid (5 *) 16l

DNTP mixture (10mM) 1.6l

Second chain enzyme mixed solution (20 *) 4l

Cumulative volume 80 μ l, gently mixing and centrifugal a little after, bathed 1.5 hours in 16 ℃ of temperature.

(2) go into 2 μ l (10U) T4 archaeal dna polymerases, 16 ℃ were reacted 45 minutes behind the mixing.

(3) add 4 μ lEDTA/Glycogen mixed solutions to stop second chain synthesis reaction.

C, purifying cDNA

(1) add 100 μ l phenol: chloroform: primary isoamyl alcohol (25: 24: 1), behind the thorough mixing 10,000rpm is in 4 ℃ of centrifugal 10min.

(2) upper water is moved in the new pipe mutually, add the 100l chloroform: primary isoamyl alcohol (24: 1), behind the vibration mixing 10, the centrifugal 10min of 000rpm.

(3) upper water is moved in the new pipe mutually, add 4M ammonium acetate and 2.5 times of volume ethanol of 1/2 volume, behind the mixing 13, the centrifugal 20min of 000rpm room temperature is with the double-stranded cDNA of precipitation synthetic.

(4) with 300 μ l80% ethanol centrifuge washings precipitation, behind the air drying 10min, with 10l sterilized water dissolution precipitation ,-20 ℃ of preservations are standby.

D, joint connect

(1) in the 0.5ml Eppendorf tube, add following reactants:

Double-stranded cDNA 5l

Marathon cDNA joint (10M) 2l

5 * DNA connects damping fluid 2l

T4 dna ligase (1U/l) 1l

Cumulative volume is 10 μ l, and 16 ℃ of connections are spent the night behind the mixing.

Behind (2) 70 ℃ of processing 10min deactivation ligase enzymes, be diluted to 50X, 250X with Tricine/EDTA, standby in-20 ℃ of preservations.

So far, be built into the double-stranded cDNA of the cotton fiber library that joint is contained at a two ends.

Embodiment 3, with 5 '-RACE-PCR amplification total length cotton RGP gene

3 ' RGP sequence according to FDD obtains designs primer behind termination codon, carry out 5 '-RACE-PCR with the Marathon cDNA amplification kit of Clontech company, amplifies total length cotton fiber RGP gene.

Its concrete steps are as follows:

1, following reactants is added in the 0.5ml PCR reaction tubes:

10 * PCR damping fluid 5l

MgCl ₂(25mM) 5l

dNTPs(10mM) 1l

ExTaq(2.5U/μl) 1l

Cotton cDNA library (50X) 5l

Synthetic primer (20pmol/ μ l) 1l

Joint primer AP1 (20pmol/ μ l) 1l

ddH ₂O 31l

Cumulative volume is 50 μ l.

Behind the mixing, carry out pcr amplification by follow procedure:

94℃4min；

94 ℃ of 30s; 65 ℃ of 45s; 72 ℃ of 2min (35 circulations);

72℃?7min；

4℃。

2,5 ' RACE-PCR product agarose gel electrophoresis result is shown in Fig. 4-A, wherein, and 1:DL2000Marker; The 2:RACE-PCR product, as seen from the figure, the RACE-PCR product presents 1 band, the about 1.2Kb of molecular weight.

3, with after the Wizard pcr amplified dna purification kit separation and purification of PCR product with Promega company, the clone advances on the pGEM-T easy Vector of Promega company, through transformed into escherichia coli JM109, extract plasmid, shown in Fig. 4-B, enzyme is cut to identify to contain and is inserted segmental mono-clonal, should clone called after pGEMF12f7, wherein, 3: λ-DNA Hind III/EcoR I marker; The EcoR I enzyme of 4:pGEM-T easy-GhRGP1 is cut.

PGEMF12f7 is carried out two-way order-checking with terminal cessation method, and its result shows that this sheet segment length is 1331bp, contains a complete reading open frame 1080bp, 359 amino acid of encoding, the overlapping 1114bp of 3 ' fragment that is obtained with FDD.This gene and known other plant RGP albumen have higher homology, show the cotton RGP gene that obtains the total length reading frame.

After the cotton RGP cDNA sequence that the fragment and 5 ' of FDD-RACE-PCR is obtained the total length reading frame merges, obtained the full length cDNA sequence of cotton RGP, 1331bp, 359 amino-acid residues of the ORF of 1080bp coding wherein, 3 ' and 5 ' non-coding region is respectively 50bp and 201bp, and its polyadenous glycosidation signal AAUAA is positioned at 103bp place, terminator codon downstream.

Embodiment 4, cotton RGP efficiently expressing in cotton fiber cell

With the expression pattern of Northern hybridization analysis GhRGP1 in the fiber growth course, the result shows that the expression of GhRGP1 strictly is subjected to the regulation and control of etap, 2 transcript accumulation peaks (shown in Fig. 3-B) occur.First peak appears at 15 days (having increased by 6 times) of primary wall top speed elongation, and second peak appears at 40 days (having increased by 7 times) that secondary wall thickens the phase, and expresses minimum (25 days time descended 12 times) at primary wall and secondary wall overlap period.

The above results shows that the distribution of GhRGP1mRNA in cotton tissue has tangible tissue specificity.The quick growth of cell is to be precursor with synthetic a large amount of cell wall polysaccharides, and the formation of the primary wall of cotton fibre is exactly a process of elongation fast, the plain polysaccharide of main synthesizing non fiber.Known xyloglucan is the main component of cotton fibre hemicellulose, and it is the highest at 15 days content, and this moment, GhRGP1 mRNA accumulation volume was also maximum, and promptly a large amount of GhRGP1 in fiber expressed consistent with synthesizing in a large number of xyloglucan.Yet the expression characteristic of GhRGP1 and cellulosic synthetic inconsistent with accumulation pattern, it may be relevant with the biosynthesizing of fiber internal layer cell walls in the great expression that thickens the phase.Hint GhRGP1 has participated in the biosynthesizing of non-cellulosic polysaccharide.

Embodiment 5, GhRGP1 efficiently expressing in intestinal bacteria

In order to show the encoding function of GhRGP1 gene, between the SphI and KpnI site of the pQE30 of Qiagen company, and transformed into escherichia coli M15 has obtained to efficiently express with the GhRGP1 gene clone in the present invention.

The present invention utilizes the multiple clone site of the pQE30 efficient expression vector of Qiagen company to realize efficiently expressing of GhRGP1.The product that the pQE30 expression vector gives expression to is the fusion rotein of additional 6 the continuous His of 5 ' end, and these 6 successive His have constituted the site of Ni-NTA gel specific combination, therefore can utilize affinity chromatography to come the purifying expression product.Design and synthesize a pair of special primer, go out the gene coded sequence that two ends have specific restriction enzyme site by molecular cloning normal experiment program with pcr amplification, gene is with after carrier is cut with identical enzyme (SphI and KpnI) enzyme, connect again, to connect product transformed into escherichia coli M15 bacterial strain, and with the LB plate screening recon that contains 100 μ g/ml penbritins and 25g/ml kantlex.After plasmid enzyme restriction evaluation and PCR evaluation, the correct recon of connection that sifts out is checked order, prove that the reading frame of the dna sequence dna that inserts carrier is correct.Have the GhRGP1 expression carrier being built into, called after pQE-30-GhRGP1 (as shown in Figure 5) is used for the abduction delivering analysis.

With screening and through identifying the recon of confirming, be inoculated in 5ml LB (containing 100g/ml penbritin and the 25g/ml kantlex) substratum, 37 ℃ of overnight incubation are inoculated in the 4ml LB nutrient solution in 1: 10 ratio then, and 37 ℃ are continued to be cultured to A ₆₀₀=0.6～0.9, the IPTG solution (the IPTG final concentration is 1mM) that adds 8l 1M, induce and get the centrifugal collection thalline of 1ml bacterium liquid after 3 hours, extract the cell soluble proteins by the method that Qiagen provides, carry out the 12.5%SDS-PAGE electrophoresis detection, the result as shown in Figure 6, wherein, 1: the M15 of no expression vector; 2: the M15 that contains pQE30; 3: contain the M15 of pQE-30-GhRGP1 but do not induce; 4,5,6 and 7 are respectively the M15 that contains pQE-30-GhRGP1 induces 1,2,3 and 4 hours.As can be seen from the figure, the GhRGP1 gene is efficiently expressed at M15.

Sequence table

<160>2

<170>

<210>1

<211>1331

<212>DNA

<213>Gossypium?hirsutum

＜214〉cotton belongs to upland cotton

<400>1

cttccatccc?ctctccctgc?tttgctctga?aaagtccaag?tttttctcca?atggcgaccg 60

tctctccaac?tcctctgttg?aaagatgagc?tcgacatcgt?gattcccacg?attaggaact 120

tggacttttt?ggagatgtgg?agaccctttt?tccagcctta?ccatctcatc?attgttcaag 180

atggtgatcc?taccaagacc?atcaaggtcc?ctgaaggctt?cgattatgag?ctttacaata 240

ggaacgacat?caacagga tt?ttgggtccta?aggcttcttg?tatctctttc?aaggattctg 300

cttgtaggtg?ctttgggtac?atggtttcca?agaagaaata?catctatacc?attgatgatg 360

actgctttgt?tgctaaagat?ccatctggca?aagacatcaa?tgcattggaa?cagcacatac 420

agaacctact?gagtccatcc?actccatttt?tctttaacac?tctttatgac?ccataccgat 480

ccggtgctga?cttcgttcgt?ggctaccctt?tcagtctccg?tgagggtgtt?accactgccg 540

tctcacacgg?tctctggctc?aacatcccgg?attatgatgc?tcccacacag?cttgtcaagc 600

cactcgagag?gaacaccagg?tatgtcgacg?ctataatgac?gattccgaag?ggaaccttgt 660

tccccatgtg?cggaatgaat?ctggcattta?accgagaatt?gatcggccct?gcaatgtact 720

ttggactcat?gggagatggt?caaccaattg?gacgatacga?tgatatgtgg?gctggctggt 780

gcaccaaggt?tatttgtgat?cacctcggat?ggggagtgaa?gaccggactt?ccctacattt 840

ggcacagcaa?agcgagcaac?ccgtttgtga?accttaagaa?agaatacaaa?ggaatttact 900

ggcaagaaga?gttgattcct?ttcttccaat?ccgttgccct?tccgaaggac?tgcaccaccg 960

tacagaaatg?ctacatcgag?atctccaagc?aagtgaaggc?gaaactcggc?aaggtcgatg 1020

actacttcaa?taagctggcg?gatgctatgg?tgacatggat?tgaagcttgg?gatgaactga 1080

acccatcggg?cgatatatcc?gccaagattc?ctaatggtgc?ttccaagtga?aaacacatca 1140

ttgtgttatt?ttgcaggttg?gagggagaac?catttatgta?tgttactaca?tatctcatag 1200

tattttatca?tatttactga?gcttagatga?acaataagaa?atttatggtt?ttatgtattg 1260

tattagactt?tgagagttag?cttctgtagt?tttaattcag?atgattgcat?ttcaattaag 1320

taaaaaaaaa?a 1331

<120>

<210>2

<211>359

<212>PRT

<213>Gossypium?hirsutum

＜214〉cotton belongs to upland cotton

<400>2

Met?Ala?Thr?Val?Ser?Pro?Thr?Pro?Leu?Leu?Lys?Asp?Glu?Leu?Asp

1 5 10 15

Ile?Val?Ile?Pro?Thr?Ile?Arg?Asn?Leu?Asp?Phe?Leu?Glu?Met?Trp

20 25 30

Arg?Pro?Phe?Phe?Gln?Pro?Tyr?His?Leu?Ile?Ile?Val?Gln?Asp?Gly

35 40 45

Asp?Pro?Thr?Lys?Thr?Ile?Lys?Val?Pro?Glu?Gly?Phe?Asp?Tyr?Glu

50 55 60

Leu?Tyr?Asn?Arg?Asn?Asp?Ile?Asn?Arg?Ile?Leu?Gly?Pro?Lys?Ala

65 70 75

Ser?Cys?Ile?Ser?Phe?Lys?Asp?Ser?Ala?Cys?Arg?Cys?Phe?Gly?Tyr

80 85 90

Met?Val?Ser?Lys?Lys?Lys?Tyr?Ile?Tyr?Thr?Ile?Asp?Asp?Asp?Cys

95 100 105

Phe?Val?Ala?Lys?Asp?Pro?Ser?Gly?Lys?Asp?Ile?Asn?Ala?Leu?Glu

110 115 120

Gln?His?Ile?Gln?Asn?Leu?Leu?Ser?Pro?Ser?Thr?Pro?Phe?Phe?Phe

125 130 135

Asn?Thr?Leu?Tyr?Asp?Pro?Tyr?Arg?Ser?Gly?Ala?Asp?Phe?Val?Arg

140 145 150

Gly?Tyr?Pro?Phe?Ser?Leu?Arg?Glu?Gly?Val?Thr?Thr?Ala?Val?Ser

155 160 165

His?Gly?Leu?Trp?Leu?Asn?Ile?Pro?Asp?Tyr?Asp?Ala?Pro?Thr?Gln

170 175 180

Leu?Val?Lys?Pro?Leu?Glu?Arg?Asn?Thr?Arg?Tyr?Val?Asp?Ala?Ile

185 190 195

Met?Thr?Ile?Pro?Lys?Gly?Thr?Leu?Phe?Pro?Met?Cys?Gly?Met?Asn

200 205 210

Leu?Ala?Phe?Asn?Arg?Glu?Leu?Ile?Gly?Pro?Ala?Met?Tyr?Phe?Gly

215 220 225

Leu?Met?Gly?Asp?Gly?Gln?Pro?Ile?Gly?Arg?Tyr?Asp?Asp?Met?Trp

230 235 240

Ala?Gly?Trp?Cys?Thr?Lys?Val?Ile?Cys?Asp?His?Leu?Gly?Trp?Gly

245 250 255

Val?Lys?Thr?Gly?Leu?Pro?Tyr?Ile?Trp?His?Ser?Lys?Ala?Ser?Asn

260 265 270

Pro?Phe?Val?Asn?Leu?Lys?Lys?Glu?Tyr?Lys?Gly?Ile?Tyr?Trp?Gln

275 280 285

Glu?Glu?Leu?Ile?Pro?Phe?Phe?Gln?Ser?Val?Ala?Leu?Pro?Lys?Asp

290 295 300

Cys?Thr?Thr?Val?Gln?Lys?Cys?Tyr?Ile?Glu?Ile?Ser?Lys?Gln?Val

305 310 315

Lys?Ala?Lys?Leu?Gly?Lys?Val?Asp?Asp?Tyr?Phe?Asn?Lys?Leu?Ala

320 325 330

Asp?Ala?Met?Val?Thr?Trp?Ile?Glu?Ala?Trp?Asp?Glu?Leu?Asn?Pro

335 340 345

Ser?Gly?Asp?Ile?Ser?Ala?Lys?Ile?Pro?Asn?Gly?Ala?Ser?Lys

350 355 359

Claims (6)

1, a kind of gene of the cotton reversible glycosylated proteinase of encoding is the nucleotide sequence with sequence 1.

2, gene according to claim 1 is characterized in that: its opening code-reading frame is for from the 51st to the 1130th totally 1080 bases.

3, the reversible glycosylated proteinase that grows cotton, the aminoacid sequence with sequence 2.

4, the carrier that contains claim 1 or 2 described genes.

5, with the vegetable cell with reversible glycosylated ability of the gene transformation of claim 1 or 2.

6, with the intestinal bacteria with reversible glycosylated ability of the gene transformation of claim 1 or 2.

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